Jiang‐Flavour Daqu (JFDQ) is a type of grain‐based fermentation starter used in Chinese liquor production. This study aimed to monitor the practical production of JFDQ and improve brewing techniques by analysing the microbial diversity and peptide profiles in JFDQ with different colours (WDQ, YDQ and BDQ) using 16S rRNA gene sequencing and peptidomics technology. A total of 110 operational taxonomic units (OTUs) were identified, with BDQ containing the most (98). Bacillus was predominant in WDQ (77.5%) and YDQ (82.3%) but decreased sharply in BDQ (29.0%). Most peptides were identified in WDQ (406), followed by YDQ (282) and BDQ (90). The content of peptides increased (7.16–9.13 g/100 g) from WDQ, YDQ to BDQ, whereas the number of peptides decreased (406–90). This phenomenon may result from a synergistic effect of the microbes in generating and consuming peptides as exogenous nitrogen sources. Additionally, six novel antimicrobial peptides were found against Escherichia coli and Staphylococcus aureus, indicating an antagonistic relationship between antimicrobial peptides and microbes in JFDQ. This study provides a credible direction for investigating the interaction between microbes and peptides in JFDQ, which could guide the practical production more scientifically.